Deoxyribonucleic acid synthetic response of mouse lymphoid cells in microculture with T-lymphocyte-dependent mitogens

Various numbers of mouse thymus, lymph node, or spleen cells were cultured in serum-containing medium with concentrations of 0.06 to 64 mug. per ml. of concanavalin A (Con A), using our version of the microculture technique. Maximal DNA synthetic responses were obtained with 1.0 million thymocytes (highest number used) at 4 mug. per ml. of Con A and with 0.75 million lymph node cells or 0.5 million spleen cells at 1 mug. per ml. of Con A. At optimal Con A concentration the magnitudes of response of 0.25 million thymus, compared with the same number of lymph node cells, were very similar. However, the response of lymph node cells was superior to that of thymocytes when 0.5 million initial cells were used, whereas 1.0 million thymocytes gave a better response than did the same number of lymph node cells. Regardless of the cell concentration, a comparison of responses by the three different cell types with the same cell number showed that the Con A concentration-DNA synthetic response curve was broadest for spleen cells, narrow for thymus cells, and intermediate for lymph node cells. Results in replicate experiments were highly reproducible, and the data indicated that peak DNA synthetic responses were not directly proportional to cell number when certain numbers of cells were used; the actual proportionality of response to cell number varied with the cell type and the mitogen concentration. When examined throughout the culture period, concentrations of Con A which were above the optimal for stimulation of DNA synthesis resulted in significant decreases in the number of viable cells. Time course experiments with Con A (or phytohemagglutinin)-stimulated BALB/c spleen cells showed that the initiation of DNA synthesis occurred between 12 and 24 hours of culture, with peak levels of 3H-thymidine incorporation occurring during the 2nd and 3rd days of culture. The presence or absence of fetal calf serum had essentially no effect on the Con A- or phytohemagglutinin-stimulated DNA synthetic responses during the 3rd day of culture. These results demonstrate that detailed evaluation of culture variables and mitogen concentrations is required for the most meaningful use of mitogen-stimulated DNA synthesis as a parameter for comparing different lymphoid cell populations.